Carboxylated heterocyclic compounds and methods of synthesis

ABSTRACT

Compositions of the present invention comprise carboxylated heterocyclic compounds, including carboxyflosequinan. The methods of the present invention also comprise the synthesis of carboxyflosequinan.

This is a divisional application of patent application Ser. No.10/281,800 filed Oct. 28, 2002 now U.S. Pat. No. 6,689,791, whichclaimed benefit, under 35 U.S.C. §119(e), to provisional applicationSer. No. 60/360,829 filed on Mar. 01, 2002 under 35 U.S.C. 111(b).

FIELD OF THE INVENTION

The present invention teaches compositions comprising carboxylatedheterocyclic compounds and the synthesis of the same.

BACKGROUND

A variety of heterocyclic compounds have been described as havingvarious pharmaceutical applications. However, the synthesis of suchcompounds, especially on a large scale, is often labor-intensive,expensive and time consuming. What is needed therefore, is a simplifiedand economical method for the synthesis and purification of heterocycliccompounds.

SUMMARY OF THE INVENTION

The present invention relates to compositions comprisingcarboxyflosequinan and the synthesis of the same.

In one embodiment, the present invention teaches a carboxylatedheterocyclic compound corresponding to3-carboxymethylsulfinyl-7-fluoro-1-methyl-4-quinolone(carboxyflosequinan) and derivatives thereof.

In one embodiment, the present invention teaches providing,3-cyanomethylthio-7-fluoro-1-methyl-4-quinolone and a first acidfollowed by the reaction of said3-cyanomethylthio-7-fluoro-1-methyl-4-quinolone and first acid underconditions such that 3-carboxymethylthio-7-fluoro-1-methyl-4-quinoloneis produced.

In another embodiment the present invention further contemplates thereaction of 3-carboxymethylthio-7-fluoro-1-methyl-4-quinolone with asecond acid and a peroxide under conditions such that3-Carboxymethylsulfinyl-7-fluoro-1-methyl-4-quinolone is produced.

DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the chemical structure of3-carboxymethylsulfinyl-7-fluoro-1-methyl-4-quinolone.

FIG. 2 displays a scheme for the synthesis of3-carboxymethylsulfinyl-7-fluoro-1-methyl-4-quinolone.

FIG. 3 depicts the results of enzyme (PKC) inhibition assays with3-carboxymethylsulfinyl-7-fluoro-1-methyl-4-quinolone.

DEFINITIONS

As used herein carboxyflosequinan refers to the chemical compositiondesignated as 3-carboxymethylsufinyl-7-fluoro-3-methyl-4-quinolonehaving the chemical structure corresponding to:

As used herein, the phrase “flosequinan” and a “a racemic mixture offlosequinan” refers to7-fluoro-1-methyl-3-(methylsulphinyl)-4(1H)-quinolinone which may alsobe described as 7-fluoro-1-methyl-3-(methylsulfinyl)-4(1H)-quinolone)and as 7-fluoro-1-methyl-3-methylsulfinyl-4-quinolone having thechemical structure of:

As used herein, “room temperature”, “RT” or “ambient temperature” isapproximately 18° C. to 25° C.

As used herein, “overnight” is approximately 8 hours, more preferably 12hours, more typically 17 hours, but can be up to approximately 30 hours.

As used herein, a “heterocyclic” compound refers to a compoundcomprising a ring composed of atoms of more than one kind.

As used herein, a “catalyst” refers to a substance that, when added to areaction mixture, changes (e.g. speeds up) the rate of attainment ofequilibrium in the system without itself undergoing a permanent chemicalchange. Examples of suitable catalysts contemplated for use in thepresent invention include, but are not limited to, antimony chloride andcarbon tetrabromide.

As used herein, a “solvent” refers to a substance that will dissolveother substances. An “organic solvent” is an organic substance that willdissolve other substances. Examples of solvents suitable for use inembodiments of the present invention include, but are not limited todichloromethane and acetonitrile.

DETAILED DESCRIPTION OF THE INVENTION

In one embodiment, the present invention describes a compositioncomprising carboxyflosequinan. In another embodiment, the presentinvention teaches methods for the synthesis of carboxyflosequinan.

The present invention also contemplates the formulation ofcarboxyflosequinan as a pharmaceutically acceptable salt. In addition,pharmaceutical formulations of carboxyflosequinan may also containbinders, fillers, carriers, preservatives, stabilizing agents,emulsifiers, buffers and excipients as, for example, pharmaceuticalgrades of mannitol, lactose, starch, magnesium stearate, sodiumsaccharin, cellulose, and magnesium carbonate. The present inventionalso contemplates the administration of carboxyflosequinan as apharmaceutically acceptable salt or formulation. The present inventionalso contemplates the administration of carboxyflosequinan andcarboxyflosequinan formulations to a subject.

Methods of producing a racemic mixture of flosequinan, as set out inU.S. Pat. Nos. 5,079,264 and 5,011,931 to MacLean et al., are herebyincorporated by reference. In one embodiment, flosequinan is preparedaccording to the protocol set out in Example 2.

Without limiting the invention to any particular mechanism,carboxyflosequinan is an enzyme inhibitors. In a specific example,carboxyflosequinan inhibit protein kinase C (herein after PKC). Thiseffect of carboxyflosequinan on enzyme activity, more particularly onPKC, has utility in therapeutics.

Experimental

The following examples serve to illustrate certain preferred embodimentsand aspects of the present invention and are not to be construed aslimiting the scope thereof.

In the experimental disclosure which follows, the followingabbreviations apply: eq (equivalents); M (Molar); μM (micromolar); N(Normal); mol (moles); mmol (millimoles); μmol (micromoles); nmol(nanomoles); g (grams); mg (milligrams); L (liters); ml (milliliters); °C. (degrees Centigrade).

All bracketed numbers [e.g. “(1)”] after the chemical name of acompound, refer to the corresponding chemical structure as designated bythe same bracketed number in FIG. 1.

All NMR spectra were recorded using Varian-Gemini 300 MHz Spectrometer.

In Examples 1, unless otherwise stated, the source for the chemicalreagents was Aldrich, Milwaukee, Wis., USA (unless a reagent wassynthesized de novo, as described in the examples). Flosequinan wassynthesized according to the protocol provided in Example 2, unlessspecified otherwise.

EXAMPLE 1 Synthesis of3-Carboxymethylsulfinyl-7-fluoro-1-methyl-4-quinolone (5)

To an efficiently stirred and gently cooled with dry-ice acetone mixtureof 12 ml of thionyl chloride (SOCl₂) and 3 ml of pyridine (Py) at −3° C.was added flosequinan (3.59 g, 15 mmol) (1) in a few portions over aperiod of approximately 1 min. During that time cooling was applied tokeep the temperature in the range 0-6° C. The mixture was stirred at 0°C. for 5 min, cooled to −5° C. and poured as a thin stream into 350 mlof ice-water with efficient stirring. After 10 min stirring at 0° C. asolid was filtered off, washed with water, and dried over phosphoruspentoxide under high vacuum. Yield 2.82 g (74%) of a crude product thatwas ˜95% pure by ¹H NMR. The crude product(3-Chloromethylthio-7-fluoro-1-methyl-4-quinolone) (2) was used in thenext step without further purification.

To efficiently stirred suspension of sodium cyanide (490 mg, 10 mmol) indry DMSO (15 ml) at room temperature under a N₂ atmosphere was addedcrude 3-chloromethylthio-7-fluoro-1-methyl-4-quinolone (1.031 g, 4 mmol)(2) in a few portions. The mixture was stirred for 1h and poured intodiluted H₂SO₄ with ice. The solid was filtered off. The filtrate wasextracted twice with ethyl acetate, the combined extracts were driedover anhydrous Na₂SO₄ and concentrated. The residue was combined withthe solid and was chromatographed on silica gel with hexane-ethylacetate (gradient 2:1,1:1,1:2) to give 644 mg (65%) of the product as abrownish solid. The product(3-Cyanomethylthio-7-fluoro-1-methyl-4-quinolone; 575 mg) (3) wasfurther purified by recrystallization from methanol to give 382 mg ofbrownish crystals.

A mixture of 3-cyanomethylthio-7-fluoro-1-methyl-4-quinolone (265 mg,1.067 mmol) (3) and 3N hydrochloric acid (8 ml) was refluxed under a N₂atmosphere for 2.5 h. The hot mixture was diluted with water (1 ml) andallowed to cool to room temperature. A solid that precipitated wasfiltered off and dried under high vacuum. The yield of3-Carboxymethylthio-7-fluoro-1-methyl-4-quinolone (4) was 270 mg(94.7%).

50% hydrogen peroxide (57 ml, 33.6 mg, 0.988 mmol) was added to asolution of 3-carboxymethylthio-7-fluoro-1-methyl-4-quinolone (4) inacetic acid (3.6 ml) at 60° C. and the mixture was stirred at 55° C. for4 h. The hot mixture was diluted with water (12 ml) and cooled to 0° C.A white solid that precipitated was filtered off and dried under highvacuum. The yield of3-Carboxymethylsulfinyl-7-fluoro-1-methyl-4-quinolone was 185 mg (72.7%)(5).

EXAMPLE 2

In this example flosequinan is prepared according to the followingprotocol:

A. Preparation of Flosequinan

i. Step I

In a clean and dry 12 L glass reactor equipped with a back suction trapplus a NaOH (25%) trap at the outlet and a back suction trap in theinlet, 3.840 L of toluene were charged and cooled to −45° C. using a dryice-acetone bath. Using appropriate safety precautions, 832 g ofphosgene were then passed through the cold toluene while stirring toprepare a 20% (wt/wt) solution. The addition of the phosgene tookapproximately 3.5 hours.

Separately, into a clean and dry 22 L glass reactor equipped with theabove-described types of back suction traps, 399 g of starting material(formula I):

was added with stirring to 4.37 L of deionized water. A separate 6.8%solution of sodium carbonate in water was also prepared by adding 297 gof sodium carbonate to 4.37 L of deionized water. Using a clean additionfunnel, the sodium carbonate solution was then slowly added withstirring to the suspension of the starting material, to create abrown-colored solution.

In preparation for the reaction step, the phosgene solution was warmedfrom −45° C. to −15° C. and the mixture of the starting material and thesodium carbonate was cooled to 10° C. The phosgene solution was thenadded over approximately 1.5 hours with stirring to the brown solution.The reaction mixture was stirred overnight allowing the desiredintermediate-A (formula II):

to precipitate out. A sample was removed for NMR assessment and theprecipitate was filtered on a 4 L sintered glass funnel. The filtratewas washed with 2×500 ml aliquots of cold deionized water and driedunder a vacuum at approximately 50° C. for 16 hours.

A 93.4% lot yield of 435 g of intermediate-A (formula II) was obtained.This procedure was repeated three more times, starting withapproximately 400 g of starting material each time. Lot yields of 448 g(94.5%), 449 g (95.9%), and 459 g (96.8%) were obtained.

ii. Step II

In a 22 L oven dried glass reactor equipped with a reflex condenser,addition funnel and temperature recorder, 11.40 L of anhydroustetrahydrofuran (THF) were added under nitrogen. To this reactor werealso added 409 g of 60% sodium hydride in oil. Eight approximately equalportions of intermediate-A (formula II) were then added to the reactor,totaling 883 g altogether. As this reaction is exothermic, care wastaken to avoid excessive heat and bubbling. Final temperature was 40°C., with a maximum observed temperature of 41° C. The reaction mixturewas stirred until hydrogen gas evolution ceased.

To the reaction mixture was then slowly added 575 ml (766.4 g) ofdimethyl sulfate, keeping the temperature below 50° C. Upon completion,the reaction mixture was stirred at 50° C. for 3 hours with the refluxcondenser on. A sample was removed for NMR assessment, and the heat wasturned off before stirring overnight.

In the morning, the stirring was stopped and the clear liquid on top wassiphoned off. This liquid was filtered using a 2-3 inch thick Celite padin a 2 L sintered glass funnel. The residue cake was kept covered tominimize contact with atmospheric moisture. The residue was collectedand washed with 4 aliquots of anhydrous THF. The filtrate and thewashings were evaporated to dryness using a rotary evaporator and theresidue obtained was dried under vacuum at approximately 36-38° C.overnight. A sample was removed for NMR assessment of the amount ofunreacted dimethyl sulfate present. The dried residue was then added to1600 ml of a 1:3 toluene:hexane mixture and vigorously stirred. Thismixture was then filtered and washed with 2×700 ml washings of 1:3toluene:hexane mixture. A reference sample was removed for NMRassessment and the residue was dried at 51-50° C. under vacuum for 36hours.

This batch yielded 871 g of intermediate-B (formula III):

for a lot yield of 91.6%. Another 907.1 g of intermediate-A wassubjected to the procedure of step II, in which the amounts of reactantsand solvents was proportionately adjusted with a yield of 850 g (87%).iii. Step III

In an oven dried 12 L glass reactor equipped with a stirrer, temperaturerecorder and addition funnel, 2550 ml of anhydrous toluene was addedunder nitrogen. Then 236 g of 60% sodium hydride in oil was added, allat room temperature. The reaction mixture was heated with continuousstirring to 75° C. using a heating mantel. Then 1.59 L of anhydrousdimethyl sulfoxide (DMSO) were added slowly and carefully over 45minutes taking care to avoid excessive bubbling. The reaction mixturewas stirred for one hour at 70-72° C. until clear and hydrogen gasevolution ceased. The heating mantel was turned off and a water bath wasused to cool the reaction mixture to 30° C.

To this mixture, 538.2 g of dry intermediate-B (formula III) was addedslowly in portions, keeping the temperature no higher than 35° C. Then1.9 L of anhydrous DMSO was added, again keeping the temperature nohigher than 35° C. The reaction mixture was stirred under nitrogen forone hour, allowing the mixture to cool to 26°. The reaction mixture wasthen quenched slowly and carefully with 320 ml of methanol. Theresulting suspension was then added slowly and with vigorous stirring toa 22 L reaction vessel containing 12.760 L of diethyl ether.

After stirring was stopped, the upper ether layer was siphoned off andthe brown oil lower layer was washed with 520 ml of fresh ether. Theoily yellow residue was triturated with 2600 ml of deionized water untila yellow precipitate formed. This precipitate was filtered using a 2 Lsintered glass funnel and the solid residue was washed with threealiquots of 130 ml cold deionized water. A reference sample was taken toassess the residue. The residue was dried under vacuum at 50-53° C. for23 hours.

This procedure produced 243 g of intermediate C (formula IV):

which represents a 38.4% yield. Two other batches of intermediate-B weretreated according to this Step III procedure, with proportionateadjustments to the amounts of reactants and solvents. The firstadditional batch of 538.2 g intermediate-B produced a 192 g (30.4%)yield, and the second additional batch of 87.38 g of intermediate-Bproduced a yield of 42 g (40.9%).iv. Step IV

In a 12 L oven dry glass reactor equipped with a stirrer, temperaturerecorder and addition funnel which has been dried by nitrogen flow for30 minutes the following chemicals were charged: 7.990 L of triethylorthoformate; 696 g of intermediate-C; 324 ml of piperdine; and 296 mlof acetic acid. The reaction mixture was heated under nitrogen to refluxat approximately 105° C. for 2 hours. A sample was removed to assess theprogress of the reaction step by NMR.

Using a water bath, the reaction mixture was then cooled to roomtemperature and stirred for 30 minutes. The final product precipitatedout and was collected by filtration on a 4 L sintered glass funnel. Theresidue was washed with 3×700 ml aliquots of diethyl ether, and a samplewas removed for NMR assessment. The residue was dried under vacuum at50-51° C. for 17 hours. A sample of the dried flosequinan product(formula V):

was removed for NMR assessment. 547 g (75.3%) yield of flosequinan wasobtained (an additional 47 g of product was scraped from the bottom ofthe sintered glass filter but was not included in this total yieldcalculation).

EXAMPLE 3

In this example, carboxyflosequinan was subjected to biochemical enzymeassays and radioligand binding assays to determine its percentinhibition of a variety of enzyme activities. Tamaoki and Nakano “Potentand specific inhibitors of protein kinase C of microbial origin”Biotechnology 8:732 (1990); Wilkinson et al. “Isoenzyme specificity ofbisindolymaleimides, selective inhibitors of protein kinase C” Biochem.J. 294:335 (1993); Tamaki et al. “Staurosporine, a potent inhibitor ofphospholipid/Ca++ dependent protein kinase” Biochem. Biophys. Res. Comm,135:397 (1986). A brief summary of the conditions for each assay isprovided below:

Protein Serine/Threonine Kinase PKCα: Human recombinant enzyme from Sf9insect cells was used in the assay. The substrate was 200 μg/ml histone.The reaction was incubated 10 mins at 25° C. in 20 mM Hepes, 10 mMMgCl₂, 0.1 mM CaCl₂. [³²P]histone was quantitated.

Protein Serine/Threonine Kinase PKC, non-selective: The enzyme wasobtained from rat brain and the substrate was 370 μg/ml histone. Thereaction was pre-incubated 5 min at 25° C., followed by a 15 minincubation at 25° C. in a buffer of 20 mM Tris-HCl, 10 nM MgCl₂.H_(O)and 0.1 mM CaCl₂.2H_(O), pH 7.4. [³²P]histone was quantitated.

FIG. 3 projects data for carboxyflosequinan in the assays describedabove. In these protein kinase assays, carboxyflosequinan was used invarying concentrations (in 1% DMSO as the vehicle). For the proteinserine/threonine kinase (PKC, non-selective), carboxyflosequinan wastested at a concentrations of 100 μM, 300 μM, and 1000 μM. Significant(e.g., greater than 50%) inhibition was observed at all threeconcentrations of carboxyflosequinan. (See, FIG. 3).

All publications and patents mentioned in the above specification areherein incorporated by reference. Various modifications and variationsof the described invention will be apparent to those skilled in the artwithout departing from the scope and spirit of the invention. Althoughthe invention has been described in connection with specific preferredembodiments, it should be understood that the invention as claimedshould not be unduly limited to such specific embodiments. Indeed,various modifications of the described modes for carrying out theinvention which are obvious to those skilled in the art are intended tobe within the scope of the following claims.

1. A method for the synthesis of3-carboxymethylsulfinyl-7-fluoro-1-methyl-4-quinolone, comprising: a)providing: i) 3-cyanomethylthio-7-fluoro-1-methyl-4-quinolone; and ii) afirst acid; b) reacting said3-cyanomethylthio-7-fluoro-1-methyl-4-quinolone and said first acidunder conditions such that3-carboxymethylthio-7-fluoro-1-methyl-4-quinolone is produced; and c)reacting said 3-carboxymethylthio-7-fluoro-1-methyl-4-quinolone with; i)a second acid; and ii) a peroxide under conditions such that3-carboxymethylsulfinyl-7-fluoro-1-methyl-4-quinolone is produced.